Novel clinical biomarkers in blood and pleural effusion for diagnosing patients with tuberculosis distinguishing from malignant tumor

Pleural effusion (PE) is a common manifestation of tuberculosis (TB) and malignant tumors but tuberculous PE (TPE) is difficult to distinguish from malignant PE (MPE), especially by noninvasive detection indicators. This study aimed to find effective detection indices in blood and PE for differentiating TB from a malignant tumor. A total of 815 patients who were diagnosed with TB or cancer in Hubei Shiyan Taihe Hospital from 2014 to 2017 were collected. Amongst them, 717 were found to have PE by thoracoscopy. Clinical characteristics, patients’ blood parameters and PE indicator information were summarized for analysis. Patients with MPE had higher percentages to be bloody and negative of Rivalta test in PE than those with TPE. For clinical indicators, comparison of the specific parameters in blood showed that 18 indicators were higher in the TPE group than in the MPE group. By contrast, 12 indicators were higher in the MPE group than in the TPE group (P < .01). In addition, in PE tests, 3 parameters were higher in the TPE group, whereas other 4 parameters were higher in the MPE group (P < .01). Then, for clinical diagnosing practice, ROC analysis and principal component analysis were applied. The top 6 relevant indicators with area under curve over 0.70 were screened out as follows: hydrothorax adenosine dehydrogenase (pADA, 0.90), hydrothorax high-sensitivity C reactive protein (0.79), percentage of blood monocyte (sMONp, 0.75), blood high-sensitivity C reactive protein (sHsCRP, 0.73), erythrocyte sedimentation rate (0.71) and blood D-dimer (0.70). Moreover, logistic regression model revealed that a specific combination of 3 biomarkers, namely, pADA, sMONp and sHsCRP, could enhance the distinguishment of TB from malignant tumor with PE (area under curve = 0.944, 95% confidence interval = 0.925–0.964). The diagnostic function of the top single marker pADA in patients from different groups was analyzed and it was found to maintain high specificity and sensitivity. The 6 indicators, namely, pADA, hydrothorax high-sensitivity C reactive protein, sMONp, sHsCRP, sESR and blood D-dimer, showed significant diagnostic value for clinicians. Further, the combination of pADA, sMONp and sHsCRP has high accuracy for differential diagnosis for the first time. Most interestingly, the single marker pADA maintained high specificity and sensitivity in patients with different statuses and thus has great value for rapid and accurate diagnosis of suspected cases.


Introduction
Pleural effusion (PE) is mainly seen in various types of inflammation, tuberculosis (TB), and malignant tumors. [1][2][3] The onset of tuberculous PE (TPE) is more insidious, [4] with slow course and lack of specificity. [5] Malignant PE (MPE) is also common, with approximately 20% being the first symptom and 30% to 40% occurring in the course of the disease, indicating poor prognosis and short survival. [6] Patients with MPE had worse prognosis than those without MPE (median survival of 7.49 vs 12.65 months, P < .001). [6] Early diagnosis and treatment could lead to enhanced prognosis.
In the current clinical practice, isolation of mycobacterium TB in the pleural fluid is difficult and could be negative in the acute setting. [7] However, the main obstacle in diagnosing malignant effusions is the presence of false-negative cytological results in approximately 40% of cases. [8] More invasive procedures (such as pleural biopsy) to identify caseating granuloma from the parietal pleura may be required. Thoracoscopic surgery is decisive for TPE and MPE [9] but it is not widely used because of its invasive property. Consequently, the development of noninvasive methods is important to differentially diagnose these 2 diseases.
Some noninvasive studies identified patients with TPE from those with malignant tumor. [10][11][12] For instance, serum total protein, albumin and globulin were significantly higher in the TB group than in the lung cancer (CA) group, whilst serum lactate dehydrogenase (sLDH) was higher in the lung CA group than in the TB group (P < .01). [13] Some researchers found that the serum D-dimer level of patients with TPE was higher than those of patients with MPE. [14] In addition, lymphocytes and macrophages were the predominant nucleated cell in MPE and TPE was characterized by a large percentage of leukocytes and lymphocytes (P < .01). [15] However, these results all meet the problems of low sensitivity and low specificity. In the present study, clinical data of 717 patients with TPE or MPE were investigated to analyze their clinical characteristics, hydrothorax parameters and blood parameters. This study has a large sample size. The significant indicators in the differential diagnosis were preliminarily expounded, which is beneficial to distinguish PE early and improve the accuracy of diagnosis.

Data collection
This research was a retrospective study. A total of 815 patients diagnosed with TB or CA in Hubei Shiyan Taihe Hospital between 2014 and 2017 were recruited in this study. All patients agreed to participate in the study and signed informed consent forms. They underwent thoracoscopic examination, and the results showed that 717 patients had PE. The age and gender characteristics of patients were shown in Table 1.
The inclusion criteria for TPE were as follows [16] : pathological examination revealed TB foci; positive for acid-fast staining or positive for the culture of mycobacterium TB and significant absorption of PE in anti-TB treatment. At least 1 of the above criteria should be met. The inclusion criteria for cancerous PE were as follows [17] : imaging examination showed thoracic mass shadow; PE was exudative; negative for acid-fast staining or negative for TB bacillus culture and the histological or cytological examination confirmed malignant tumor. All the above criteria must be met.

PE and blood statistical analysis
All data below were collected from Hubei Shiyan Taihe Hospital.
Numerical analysis results showed that the data obeyed the normal distribution. SPSS 18.0 software was used for statistical analysis of count data by chi-square test and measurement data by independent t test. Comparison was analyzed using analysis of variance between groups. The ROC curve was used to determine the best threshold (cut off) and the area under curve (AUC) after series-parallel experiment. P < .05 was considered statistically significant.

Demographic and clinical characteristics of study population
A total of 717 patients with TPE or MPE were recruited in this study. These patients were divided into 2 groups: MPE (147, 20.4%) and TPE (570, 79.6%). As shown in Table 2, multiple clinical status of patients and physical characteristics of the patient's PE was analyzed to distinguish TPE from MPE. Amongst patients with hydrothorax, those with TB had a higher possibility to experience fever. Besides, patients with malignant tumor (40.0%) were more likely to have bloody PE than those with TB. The PE of patients with CA (67.3%) had a higher percentage to be turbid. Amongst patients with hydrothorax, those with TB (91.6%) had a higher percentage to be positive in Rivalta test (Table 2).

Discriminative indicators in clinical practice to identify TPE and MPE
The indicators in the blood and PE samples were examined to observe the clinical features of TPE and MPE. Patients with TB and tumor were separated using unsupervised hierarchical clustering, with heatmap shown in Figure 1. The results showed that the serum and PE indicators of patients with tumor compared with those of patients with TB are quite different (Fig. 1).
Blood parameters and PE indicators were compared and analyzed through Mann-Whitney U test to further obtain effective identification indicators (as shown in Table 3 and Fig. 2 Fig. 2 and Table 3).

Effective markers to distinguish tuberculosis and malignant tumor with PE
The 37 indicators were applied to construct ROC curves to further screen effective diagnostic indicators. On the basis of the area AUC, sensitivity, and specificity), top 6 indicators with high diagnostic value were screened out (AUC ≥ 0.700; pADA, pHsCRP, sMONp, sHsCRP, sESR and sD-dimer; Fig. 3 and Table 4). In addition, principal component analysis (PCA) with these 6 serum or PE indicators revealed a clear separation between TB and malignant tumor (Fig. 4). Comparison between TB and tumor revealed that the pADA in PE showed the best AUC of 0.90 [95% confidence interval (CI): 0.87-0.93]. The potential combination schemes of metabolic biomarkers based on logistic regression analysis were applied to enhance the sensitivity and accuracy of diagnosis of TB from malignant tumor with PE. As shown in Figure 5, the combination of 3 markers (pADA, sMONp and sHsCRP) remarkably enhanced the AUC to 0.944 (95% CI: 0.925-0.964). These results indicated that the 3 indicators could act as a promising combination for distinguishing TB from tumor with PE.

Diagnostic indicator of pADA for evaluation in different clinical patients' characteristics
According to the above, the best indicator, namely, pADA, was close to the value of the combination of 3 markers (the only 1 ≥ 0.9). Thus, the alteration of indicators in different characteristics of patients was of great interest. In the stratified analysis, the AUC values of the pADA of males and females were 0.897 and 0.910. The AUC of patients with fever was 0.932, whilst that of patients without fever was 0.894, suggesting that the diagnosis accuracy of fever patients was higher. The AUC of age < 25 was 0.932, 25 ≤ age < 45 was 0.939, 45 ≤ age < 65 was 0.889 and age ≥ 65 was 0.868. Although with the increase in age, the AUC showed a downward trend and the diagnostic accuracy decreased (Fig. 6). The indicator pADA still showed the continuous distinguishing function as a diagnostic marker for TB and tumor with PE.

Table3
(Continued) www.md-journal.com The percentage of positive Rivalta test for TPE was higher than that for MPE (P < .01). A study reported that the positive rate of Rivalta test is parallel to the amount of total protein in body cavity effusion. [23] Some researchers showed that the protein level in TPE is higher than that in MPE (P < .05). [24] Consistently, the present results showed that the blood total protein in TPE was higher than that in MPE (69.09 ± 1.72 vs 64.01 ± 0.67, P = .15), which could explain that the percentage of positive Rivalta test for TPE was higher than that for MPE.
In this study, serum D-dimer was higher in the TPE group than in the MPE group (2.69 ± 1.19 vs 0.67 ± 0.07, P = .37). Other researchers proved that the serum D-dimer level of patients with TPE was higher that of patients with MPE. [14] However, they found that the difference of D-dimer in PE was more obvious. [14] These findings showed that D-dimer is a highly sensitive index in serum and PE, thus helpful for identifying TPE and MPE. sLDH was higher in the MPE group than in the TPE group (221.54 ± 31.01 vs 170.62 ± 6.02, P = .12). sLDH level was reported to be positively correlated with lymphoma-associated malignant PE (L-MPE, OR: 1.005, 95% CI: 1.003-1.007, P < .001). In addition, sLDH > 460 U/L distinguished L-MPE from TPE, with a sensitivity of 76% and a specificity of 81%. [20] Consistent with the present results, other researchers observed that the sLDH in MPE was higher than that in TPE (P = .08). [25] In multivariate logistic regression analysis, the ratio of sLDH to pleural fluid lymphocyte count was positively correlated with MPE. The sensitivity and specificity of this ratio were 0.63 (95% CI: 0.51-0.73) and 0.85 (95% CI: 0.68-0.94), respectively. [25] Therefore, sLDH is an important indicator for distinguishing TPE from MPE.
High sensitivity C reactive protein (HsCRP) is widely used as a sensitive but nonspecific marker of systemic inflammation. [26,27] Increased sHsCRP levels have been reported in many lung diseases, including tumors and TB. [28,29] In the present study, the median levels of pHsCRP and sHsCRP were higher in the TPE group than in the MPE group (24.63 ± 1.16 vs 8.07 ± 0.87 and 50.30 ± 2.67 vs 19.25 ± 3.20, P < .01). The AUC values of pHsCRP and sHsCRP were 0.79 and 0.73, respectively. Consequently, HsCRP is an important reference indicator to differentiate TPE from MPE. A meta-analysis showed that the optimal critical value of pHsCPR was 21.9 mg/dL; the values above the critical value were classified as TPE and those below the critical value were classified as MPE, the sensitivity was 0.91 (0.73-0.98) and the specificity was 0.82 (0.7-0.9). [30] Although HsCRP is a valuable diagnostic indicator, the diagnosis efficiency is low. Thus, the choice of multi-index joint analysis is conducive to improving the diagnosis efficiency and accuracy. Through logical analysis, 6 relevant indicators (pADA, pHsCRP, sD-dimer, sESR, sHsCRP and sMONp) were selected. The logistic regression model showed that 3 variables of pADA, sMONp and sHsCRP could better help distinguish patients with PE by TB from malignant tumor. The combined AUC of the 3 factors could reach 0.94 (95% CI: 0.91-0.97), higher than that of any single index. Therefore, they have great significance for the clinical differentiation between TPE and MPE. In agreement with the present study, 1 study analyzed 118 patients, including 84 patients with MPE (71.2%) and 34 patients with TPE (28.8%). The results showed that the pADA of TPE was higher than that of MPE (P < .05). [25] Moreover, others have proven that elevated levels of sHsCRP and pADA in PE were useful in distinguishing TPE from MPE. [31,32] However, only 1 study had a different result. After analyzing 17 patients with L-MPE and 216 patients with TPE, the authors found no statistically significant difference in sHsCRP and pADA levels between the 2 groups, [20] and the reason could be related to the number of patients with MPE included in this study.
At present, to achieve enhanced treatment efficacy in clinical practice, many researchers were interested in exploring the differentiation between TPE and MPE. [33][34][35][36] Some of them focused on inflammatory factors. The biomarkers of PE in 22 patients with MPE and 5 patients with TPE were compared. IL-1, IP-10, IL-13, and IFN-γ were significantly higher in TPE (P < .05). The  . CA= tumor, pADA = hydrothorax adenosine dehydrogenase, pHsCRP = hydrothorax high sensitivity C reactive protein, sD-dimer = blood D-dimer, sESR = erythrocyte sedimentation rate, sHsCRP = blood high sensitivity C reactive protein, sMONp = percentage of blood monocyte, TB = tuberculosis. level of basic fibroblast growth factor in MPE was higher than that in TPE (P < .05). [33] The highest AUC was found in IP-10 (AUC = 0.95, 95% CI, P < .01), followed by IL-13 (AUC = 0.86, 95% CI, P < .05). [33] However, though 1 of the indicators in this study showed a high AUC, detection is not a common clinical indicator, and it is complicated. The sample size was also small, the reliability was weak, and performing stratified analysis was difficult. Another study found that the fibronectin and cathepsin G in patients with MPE were significantly higher than those in patients with TPE, whilst leukotriene-a4 hydrolase was lower than in patients with TPE. [34] The AUC was determined to be 0.285 for fibronectin (95% CI: 0.174-0.396), 0.64 for leukotriene-a4 hydrolase (95% CI: 0.518-0.762), 0.337 for cathepsin G (95% CI: 0.218-0.456), and 0.793 for a combination of these candidate markers (95% CI: 0.697-0.888). The AUC was significantly lower than that in the present study. In this study, the results exhibited more significant advantages of high diagnostic accuracy (high AUC, high sensitivity and specificity) and large sample size, which indicate high data reliability. In addition, pADA, sHsCRP and sMONp are all clinically common and easy-to-collect specimens, which are convenient and cheap to test. Thus, they do not pose additional burden on patients. Hierarchical analysis could be performed because of the large sample size, and the results showed that the diagnostic efficiency of pADA differed in various age groups. As age increased, the diagnostic efficiency of pADA gradually decreased. This phenomenon could be related to the percentage of TB decreasing, whilst CA diagnosis increased with age. This finding suggested that patients under 45 years could choose the single indicator pADA for diagnostic detection. Age factors also affect  changes in sMONp, and aging leads to a decrease in immune function, resulting in a decrease in the number and quality of monocytes in the blood. [37] Meanwhile, factors such as age also affect changes in sHsCRP, with significant differences between older and younger people. [38] These indicate that the diagnostic model constructed by the 3 indicators pADA, sHsCRP and sMONp, although having high diagnostic efficacy, can be influenced by various factors, and we will continue to validate and optimize the diagnostic indicators and their influencing factors in the subsequent studies. The gold standard for differentiating TPE and MPE in clinical practice still relies on pathological tissue biopsy. [39] All cases in the present study were examined by thoracoscopy, and pathological biopsy was completed in most cases, ensuring the accuracy of diagnosis. However, for some patients who refuse to accept the invasive examination or whose constitution is difficult to bear invasive examination, the effective detection index of noninvasive examination provides a strong basis for timely diagnosis and accurate treatment. Further research and exploration are worth conducting.

Conclusion
In summary, the results showed some noninvasive and valuable markers for differentiating TPE from MPE. Although the gold standard for differentiating TPE and MPE still relies on pathological tissue biopsy, for some patients who refuse to accept invasive examination or whose constitution is difficult to bear invasive examination, the effective detection index of noninvasive examination provides a strong basis for timely diagnosis and accurate treatment.